A new approach to protein modification is under investigation. Methods are being developed to incorporate substitution-inert metal ion labels into protein sites which have been designed to accomodate metal ions (as opposed to native metal ion binding sites). These methods should be applicable to a wide variety of proteins and peptides. It is proposed that substitution-inert metal ions can serve as particularly stable radiolabels (57Co(III)), paramagnetic labels (Cr(III)) and heavy metal ion labels (Ru(II)) of proteins and hormones. It has already been shown that this approach can be used to modify an active site residue in an enzyme. A particularly promising method involves chelation of the substitution-inert metal ion by an azo tyrosine or azo histidine derivative of the protein. The azo chromophore provides a convenient means of monitoring the extent and specificity of the metal ion complexation. The azo dye chelate can be produced by diazotization of a protein or peptide tyrosine or histidine followed by metal ion incorporation. Alternately it is proposed that a metal complex of an appropriately modified aromatic amine can be synthesized and then incorporated into the protein of interest by diazotization to give a covalently bound complex in which the diazotized amino acid forms part of the chelate. Since a major part of the complex is formed prior to protein binding the latter method makes it less likely that protein damage would occur during the synthesis of the stable metal ion derivative. In the course of these studies it has been found that the spectral methods used to characterize azo proteins are based on incorrectly characterized analogues. Thus, an additional objective of the proposed studies is to provide a sound basis for the characterization of azo proteins and related hormone derivatives. These studies should also establish conditions for the selective modification of histidines and tyrosines in proteins. The availability of a reliable method for characterizing azo proteins should significantly improve the value of diazotization as a useful method for protein modification.